Barrera Nelson P, Ge Haifang, Henderson Robert M, Fitzgerald William J, Edwardson J Michael
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
Micron. 2008;39(2):101-10. doi: 10.1016/j.micron.2006.12.006. Epub 2007 Jan 13.
Fast neurotransmission involves the operation of ionotropic receptors, which are multi-subunit proteins that respond to activation by opening an integral ion channel. Examples of such channels include the GABA(A) receptor, the 5-HT(3) receptor and the P2X receptor for ATP. These receptors contain more than one type of subunit, although the exact subunit stoichiometry and arrangement around the receptor rosette is often unknown. We are using atomic force microscopy (AFM) of purified receptors to address these issues. Measurement of the molecular volume of the receptor permits the determination of the number of subunits that it contains. Furthermore, analysis of the geometry of complexes between receptors and subunit-specific antibodies reveals the subunit arrangement. Our AFM-based approach has so far been dependent on manual data processing, which is both time-consuming and prone to operator bias. In this study, we set out to develop a novel method capable of automatic segmentation and quantitative analysis of both single receptor particles and receptor-antibody complexes. The method was validated using images of wild type and mutant forms of the P2X(6) receptor. We suggest that the automated method will greatly facilitate further progress in the use of AFM for the determination of receptor and multi-protein architecture.
快速神经传递涉及离子型受体的运作，这些受体是多亚基蛋白质，通过打开一个完整的离子通道对激活做出反应。这类通道的例子包括γ-氨基丁酸A（GABA(A)）受体、5-羟色胺3（5-HT(3)）受体以及ATP的P2X受体。这些受体包含不止一种类型的亚基，尽管围绕受体花环的确切亚基化学计量和排列通常并不清楚。我们正在使用纯化受体的原子力显微镜（AFM）来解决这些问题。测量受体的分子体积可以确定其所含亚基的数量。此外，对受体与亚基特异性抗体之间复合物的几何结构分析揭示了亚基的排列。我们基于AFM的方法迄今依赖于人工数据处理，这既耗时又容易出现操作者偏差。在本研究中，我们着手开发一种能够对单个受体颗粒和受体-抗体复合物进行自动分割和定量分析的新方法。该方法通过P2X(6)受体野生型和突变型的图像进行了验证。我们认为，这种自动化方法将极大地促进在利用AFM确定受体和多蛋白结构方面的进一步进展。
